Aromatic acyltransferase genes

ABSTRACT

The present invention provides a protein having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12 or a protein having a modified amino acid sequence thereof and having an activity of transferring an aromatic acyl group to a sugar residue of a flavonoid; a gene, especially cDNA, encoding the protein; and use thereof. For example, by introducing the above gene into a plant expressing hydroxycinnamate 1- O -glucosyltransferase gene, optionally together with a cDNA encoding a protein having the amino acid sequence as shown in SEQ ID NO: 14, 16 or 18 or a protein having an amino acid sequence derived therefrom by modification and having an activity of glucosylating a hydroxyl group at position 1 of hydroxycinnamic acid, and then expressing the introduced gene(s), it is possible to acylate the sugar residue of flavonoids in flowers of the plant to thereby confer a blue color on the flowers.

TECHNICAL FIELD

The present invention relates to a gene encoding a protein having anactivity of transferring an aromatic acyl group to sugar residue offlavonoid using 1-O-acyl-β-D-glucose as an acyl donor; a gene encoding aprotein that has an activity of transferring a glucosyl group to ahydroxyl group at position 1 of hydroxycinnamic acid using UDP-glucoseas a glucosyl donor; and a method of using the same.

BACKGROUND ART

Plant color is one of the most important characters from an industrialviewpoint. As seen from pursuit of diversified flower colors, goodexpression of fruit colors, stabilization and uniformity of expressedcolors, and so on, plant color is a big economical factor in flowers andornamental plant, fruit trees and vegetables. Among plant pigments, themost abundantly seen are compounds generically termed anthocyanin. Cellsand tissues where anthocyanin is accumulated present various colors fromlight blue to dark red. To date, almost 500 types of anthocyanin havebeen reported from various plant species, and their colors are mainlydepending on the chemical structures thereof. Anthocyanidin (an aglyconethat is the skeleton of anthocyanin) does not exist in plant bodies asit is, but exists in a modified form which has undergone glucosylationor acylation. Through glucosylation, anthocyanidin becomes nontoxicanthocyanin and stabilized; also, it becomes water-soluble and dissolvedin cell vacuoles. A large number of glycosylated anthocyanins undergofurther modification such as glycosylation, acylation or methylation. Inparticular, acylation increases the stability of anthocyanin moleculesin vacuoles. Acylation by an aromatic acyl group bathochromically shiftsthe UV/visible absorption maximum of anthocyanins as a result ofintramolecular association of the aglycone and the aromatic acylgroup(s). Therefore, plant tissues with accumulation of acylatedanthocyanin with aromatic acyl groups present a purple to blue color inmany occasions. Anthocyanins form complex pigments throughintramolecular association with aromatic acyl groups; intermolecularassociation with co-pigments (such as acylglucose, flavone or flavonol)or metal ions; coordinate bond with metal ions; bond with polypeptides;etc. in vacuoles and presents diversified colors. Therefore, acylationof anthocyanin is one of the important chemical reactions in expandingthe diversity of flower colors with anthocyanin.

Color expression with anthocyanin, a character which can be directlyconfirmed with eyes, has become a target for a great number of genetic,biochemical and molecular biological studies. To date, genes involved inflavonoid including anthocyanin biosynthesis have been cloned fromfloricultural plants and experimental model plants such as petunia(Petunia x hybrida), snapdragon (Antirrhinum majus), morning glory(Pharbitis nil or Ipomoea nil), Arabidopsis thaliana; fruits such asapple (Malus x domestica), grape (Vitis vinifera); vegetables such asegg plant (Solanum melongena), perilla (Perilla frutescens); and so on.The mechanism of flower color expression with anthocyanin is now beingelucidated by analysis by means of natural product chemistry andphysiology.

Difference in color expression via accumulation of anthocyanin in plantspecies such as gentian (Gentiana spp.), prairie gentian (Eustomagrandiflorum), morning glory, lobelia (Lobelia erinus), verbena (Verbenax hybrida) or cineraria (Senecio cruentus or Pericallis cruenta) isbasically attributable to difference in the aglycone (pelargonidin,cyanidin, delphinidin, petunidin, malvidin, etc.) of anthocyanin, and itis known that accumulation of delphinidin-type pigments is effective forblue color expression. On the other hand, difference in flower colorexpression in plants such as petunia, delphinium (Delphinium spp.) orbutterfly pea (Clitoria ternatea) is attributable to difference in thebinding pattern of sugar and acyl group to anthocyanidin and the numberof bonds thereof. Acyl groups do not directly bind to anthocyaninaglycone; in many cases they bind to sugar residues (such as glucose)bound to anthocyanidin. It is reported that the caffeoyl group (anaromatic acyl group) binding to a glucosyl group at position 3′ ofanthocyanin B ring in Gentiana triflora and the p-coumaroyl group (anaromatic acyl group) binding to glucosyl groups at positions 3′ and 5′in butterfly pea and Dianella spp. are intramolecularly associating withanthocyanin aglycone at closer positions than other aromatic acyl groupsbinding to other glucosyl groups at positions 3, 5, 7, etc. (Yoshida etal., (2000) Phytochemistry 54: 85-92; Terahara et al., (1996) Journal ofNatural Products 59: 139-144; Bloor (2001) Phytochemistry 58: 923-927).Therefore, it is reasonably presumed that modification of glucosylgroups at positions 3′ and 5′ of anthocyanin with aromatic acyl groupswill be able to express purple or blue colors in cells, tissues andorgans of plants. However, genes to be used for such a purpose have notbeen isolated yet.

With respect to acylation of anthocyanin, there have been reportedacylation with aliphatic acyl groups (such as acetyl, malyl, malonyl,methylmalonyl or succinyl) and acylation with aromatic acyl groups (suchas p-coumaroyl, caffeoyl, feruloyl, sinapoyl, p-hydroxybenzoyl orgalloyl).

As a gene encoding acylation of anthocyanin sugar residue with analiphatic acyl group, there is reported a gene encoding a protein havingan activity of transferring a malonyl group to a sugar residue atposition 3 of flavonoid using an aliphatic acyl-CoA thioester as anacyldonor in dahlia (Dahlia variabilis) (Suzuki et al., (2002) PlantPhysiology 13: 2142-2151; Japanese Unexamined Patent Publication No.2002-233381), cineraria (PCT/WO96/25500; Suzuki et al., (2003) PlantBiotechnology 20: 229-234), chrysanthemum (Dendranthema x morifolium)(Suzuki et al., (2004) Plant Science 166: 89-96) and verbena anddeadnettle (Lamium purpureum) (Suzuki et al., (2004) Journal ofMolecular Catalysis B: Enzymatic 28: 87-93). Further, a gene encoding aprotein having an activity of transferring a malonyl group to a sugarresidue at position 5 of flavonoid using aliphatic acyl-CoA thioester asan acyl donor has been reported from Salvia splendens (Suzuki et al.,(2001) Journal of Biological Chemistry 276: 49013-49019; Suzuki et al.,(2004) Plant Journal 38: 994-1003; PCT/WO01/92536); and Salviaguaranitica, lavender (Lavendula angustifolia) and perilla(PCT/JP01/04677).

Further, as a gene encoding acylation of anthocyanin sugar residue withan aromatic acyl group, there is reported a gene encoding a proteinhaving an activity of transferring an aromatic acyl group to a sugarresidue at position 3 of flavonoid using aromatic acyl-CoA thioester asan acyl donor in perilla and lavender (PCT/WO96/125500;Yonekura-Sakakibara et al., (2000) Plant Cell Physiology 41: 495-502)and petunia (PCT/WO01/72984). Still further, a gene encoding a proteinhaving an activity of transferring an aromatic acyl group to a sugarresidue at position 5 of flavonoid using an aromatic acyl-CoA thioesteras an acyl donor in Gentiana triflora (PCT/WO96/25500; Fujiwara et al.,(1998) Plant Journal 16: 421-431) and prairie gentian (Noda et al.,(2000) Breeding Research 3 (Supplement 1): 61; Noda et al., (2002) The20th Annual Meeting of the Japanese Society of Plant Cell and MolecularBiology: Abstract: 145).

Thus, those reported genes and its proteins catalyzes the acyl transferto the anthocyanin sugar residues using acyl-CoA thioester asacyl-donor. However, it is reported that acyl donors include, inaddition to acyl-CoA thioester, chlorogenic acid and 1-O-acyl-β-D-glucose (Steffens (2000) Plant Cell 12: 1253-1255).

With respect to proteins having an acyl transfer activity usingchlorogenic acid as an acyl donor, purification and biochemical analysisof chlorogenic acid:glucaric acid caffeoyltransferase(5-O-caffeoylquinic acid:glucaric acid caffeoyltransferase) from tomato(Lycopersicon esculentum) have been reported (Strack and Gross (1990)Plant Physiology 92: 41-47).

As proteins having an activity of 1-O-acyl-β-D-glucose dependentacyltransferase activity, the following reports have been made. Withrespect to choline sinapoyltransferase involved in 1-O-sinapic acidester metabolism (1-O-sinapoyl-β-D-glucose:choline1-O-sinapoyltransferase), partial purification and characterization fromseeds of wild radish (Raphanus sativus) and white mustard (Sinapis alba)(Gräwe and Strack (1986) Zeitschrift für Naturforchung 43c: 28-33);analysis of Arabidopsis thaliana mutants and cloning of the gene(Shirley et al., (2001) Plant Journal 28:83-94) and biochemical analysisusing a recombinant protein (Shirley and Chapple (2003) Journal ofBiological Chemistry 278: 19870-19877); and cloning of SNG2 gene fromBrassica napus (Milkowski et al., (2004) Plant Journal 38: 80-92) havebeen reported.

With respect to malate sinapoyltransferase involved in sinapic acidester metabolism (1-O-sinapoyl-β-D-glucose:malate1-O-sinapoyltransferase), localization in Raphanus sativus cells (Sharmaand Strack (1985) Planta 163: 563-568), measurement of activity inBrassica napus seeds and seedlings (Strack et al., (1990) Planta 180:217-219), measurement of enzyme activity in seedlings and plantlets ofArabidopsis thaliana and Brassica rapa ssp. oleifera (Mock et al.,(1992) Zeitschrift für Naturforchung 47c: 680-682), protein purificationand biochemical analysis from wild radish hypocotyls (Gräwe et al.,(1992) Planta 187: 236-241), analysis of Arabidopsis thaliana mutantsand cloning of SNG1 gene (Lehfeldt et al., (2000) Plant Cell 12:1295-1306; PCT/WO02/04614), and localization in cells of leaf tissue inArabidopsis thaliana (Hause et al., (2002) Planta 215: 26-32) have beenreported.

With respect to glucose acyltransferase involved in fatty acidmetabolism (1-O-butyryl-β-D-glucose: 1-O-butyryl-β-D-glucose2-O-butyryltransferase), measurement of enzyme activity in Lycopercsiconpennellii (Ghangas and Steffens (1995) Archives of Biochemistry andBiophysics 316: 370-377; Ghangas (1999) Phytochemistry 52: 785-792),purification and determination of partial amino acid sequences (Li etal., (1999) Plant Physiology 121:453-460) and cloning of gene (Li andSteffens (2000) PNAS 97: 6902-6907; PCT/WO97/48811) have been reported.

With respect to 1-O-indole-3-acetyl-β-D-glucose:myo-inositolindole-3-acetyltransferase involved in indoleacetic acid metabolism,measurement of enzyme activity from corn (Zea mays) (Michalczuk andBandurski (1980) Biochemical Biophysics Research Communication 93:588-592), and purification and biochemical analysis of protein andanalysis of partial amino acid sequence (Kowalczyk et al., (2003)Physiologia Plantarum 119:165-174) have been reported.

With respect to 1-O-hydroxycinnamoyl-β-D-glucose:bethanidine diglucosideO-hydroxycinnamoyltransferase involved in betalain biosynthesis,detection of activity from suspension culture cells of wild spinach(Chenopodium rubrum) or petals of Lampranthus sociorum (Bokern andStrack (1988) Planta 174:101-105; Bokern et al., (1991) Planta 184:261-270), and purification of protein and analysis of biochemicalproperties thereof (Bokern et al., (1992) Botanica Acta 105: 146-151)have been reported.

With respect to β-glucogallin (1-O-galloyl-β-D-glucose) dependentgalloyltransferase involved in gallotannin biosynthesis, purification ofprotein and analysis of biochemical properties thereof from Stag's hornsumach (Rhus typhina) leaves (Niemetz and Gross (2001) Phytochemistry58: 657-661; Fröhlich et al., (2002) Planta 216: 168-172) and Englishoak (Quercus robur) leaves (Gross et al., (1986) Journal of PlantPhysiology 126: 173-179) have been reported.

As described above, purification of proteins having an activity ofcatalyzing acyl transfer reaction using 1-O-acyl-β-D-glucose as an acyldonor; elucidation of the biochemical properties of such proteins; andcloning of genes encoding such proteins have already been reported.However, with respect to detection of the activity of1-O-acyl-β-D-glucose dependent acyltransferase that transfers an acylgroup to sugar residues of flavonoids (such as anthocyanin), there hasbeen only one report about 1-O-sinapoyl-β-D-glucose:anthocyanidintriglucoside sinapoyltransferase in cultured cells of carrot (Daucuscarota) (Glaessgen and Seitz (1992) Planta 186: 582-585). Purificationof such a protein with activity or cloning of genes encoding the samehas not been performed yet.

-   [Patent Document 1] Japanese Unexamined Patent Publication No.    2002-233381-   [Patent Document 2] PCT/WO 01/92536-   [Patent Document 3] PCT/WO 01/72984-   [Patent Document 4] PCT/WO 02/04614-   [Patent Document 5] PCT/WO 97/48811

DISCLOSURE OF THE INVENTION Problem for Solution by the Invention

It is an object of the present invention to obtain a gene encoding aprotein having an activity of transferring an acyl group to a sugarresidue of a flavonoid, preferably, a protein having an activity oftransferring an aromatic acyl group to one or more positions of a sugarresidue of a flavonoid (inducing anthocyanin) not using acyl-CoA butusing 1-O-acyl-β-D-glucose as an acyl donor. By introducing the geneobtained by the invention encoding a protein having aromatic acyltransfer activity or a gene similar thereto into a plant and expressingtherein, it is possible to modify the types of flavonoid compoundsaccumulated therein to thereby modify the plant color, such as flowercolor or fruit color. Further, by regulating gene expression by RNAimethod or the like with the gene of the invention and transferring genesencoding known modification enzymes in anthocyanin (such asglucosyltransferase, acyltransferase, methyltransferase), it is possibleto allow biosynthesis of non-inherent anthocyanins in various plantspecies to thereby create plants presenting novel colors.

Means to Solve the Problem

The present inventors have found an enzyme activity in butterfly peapetals that catalyzes a reaction transferring an aromatic acyl group toa sugar residue of anthocyanin using 1-O-acyl-β-D-glucose as an acyldonor. Then, the inventors have purified the enzyme and determined apartial amino acid sequences thereof The nucleotide sequences of genesencoding proteins that catalyze reactions using 1-O-acyl-β-D-glucose asan acyl donor are highly homologous to the nucleotide sequences of genesencoding serine carboxypeptidase (SCPase). Thus, those proteins thatcatalyze reactions using 1-O-acyl-β-D-glucose as an acyl donor arecalled serine carboxypeptidase-like acyltransferase (SCPL-AT) (Milkowskiand Strack (2004) Phytochemistry 65: 517-524). Then, the inventorssynthesized degenerate primers based on the predicted amino acidsequences and nucleotide sequences existing in common in SCPase andSCPL-AT. Using these primers, RT-PCR was performed to amplify cDNAfragments, followed by determination of the nucleotide sequences thereofBased on the resultant nucleic acid sequence information, the entireprotein-encoding region of the gene of interest was cloned by screeningof cDNA library, rapid amplification of cDNA end (RACE) and reversetranscription-polymerase chain reaction (RT-PCR). Subsequently, cDNAfragments that have all of the partial amino acid sequences of thepurified protein and homologues of the cDNA fragments were cloned. Inthe same manner, cDNA homologues were also cloned from Gentiana trifloraand lobelia. For functional analysis of resultant clones, recombinantproteins obtained with Baculovirus-insect sell recombinant proteinsproduced by the Baculocirus-insect cell expression system were used toconfirm enzyme activities. The present invention has been achieved basedon the above-described findings.

The present invention provides the following [1] to [19].

[1] The 1st aspect of the present invention relates to a gene encoding aprotein having an activity of transferring an aromatic acyl group to asugar residue of a flavonoid using 1-O-acyl-β-D-glucose as an acyldonor.

[2] The 2nd aspect of the present invention relates to the gene of [1]above, which encodes any one of the following proteins (a) to (d):

-   (a) a protein having the amino acid sequence as shown in SEQ ID NO:    2, 4, 6, 8, 10 or 12;-   (b) a protein having the amino acid sequence as shown in SEQ ID NO:    2, 4, 6, 8, 10 or 12 which has addition, deletion and/or    substitution of one or plurality of amino acids;-   (c) a protein having an amino acid sequence which shows 20% or more    homology to the amino acid sequence as shown in SEQ ID NO: 2, 4, 6,    8, 10 or 12;-   (d) a protein having an amino acid sequence which shows 70% or more    homology to the amino acid sequence as shown in SEQ ID NO: 2, 4, 6,    8, 10 or 12.    [3] The 3rd aspect of the present invention relates to a gene which    hybridizes to a part or the whole of a nucleic acid represented by    the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11,    or a nucleic acid encoding the amino acid sequence as shown in SEQ    ID NO: 2, 4, 6, 8, 10 or 12 under stringent conditions and encodes a    protein having an activity of transferring an aromatic acyl group to    a sugar residue of a flavonoid using 1-O-acyl-β-D-glucose as an acyl    donor.    [4] The 4th aspect of the present invention relates to a gene from    butterfly pea or lobelia encoding a protein that has an activity of    transferring a glucosyl group to a hydroxyl group at position 1 of    hydroxycinnamic acid using UDP-glucose as a glucosyl donor and    synthesizes an acyl donor.    [5] The 5th aspect of the present invention relates to the gene of    [4] above, which encodes any one of the following proteins (a) to    (d):-   (a) a protein having the amino acid sequence as shown in SEQ ID NO:    14, 16 or 18;-   (b) a protein having the amino acid sequence as shown in SEQ ID NO:    14, 16 or 18 which has addition, deletion and/or substitution of one    or plurality of amino acids;-   (c) a protein having an amino acid sequence which shows 20% or more    homology to the amino acid sequence as shown in SEQ ID NO: 14, 16 or    18;-   (d) a protein having an amino acid sequence which shows 70% or more    homology to the amino acid sequence as shown in SEQ ID NO: 14, 16 or    18.    [6] The 6th aspect of the present invention relates to a gene from    butterfly pea or lobelia which hybridizes to a part or the whole of    a nucleic acid represented by the nucleotide sequence as shown in    SEQ ID NO: 13, 15 or 17 or a nucleic acid encoding the amino acid    sequence as shown in SEQ ID NO: 14, 16 or 18 under stringent    conditions and encodes a protein having an activity of transferring    a glucosyl group to a hydroxyl group at position 1 of    hydroxycinnamic acid using UDP-glucose as a glucosyl donor and    synthesizing an acyl donor.    [7] The 7th aspect of the present invention relates to a vector    comprising the gene of any one of [1] to [3] above.    [8] The 8th aspect of the present invention relates to a vector    comprising the gene of any one of [4] to [6] above.    [9] The 9th aspect of the present invention relates to a host cell    which has been transformed by the vector of [7] or [8] above.    [10] The 10th aspect of the present invention relates to a protein    encoded by the gene of any one of [1] to [6] above.    [11] The 11th aspect of the present invention relates to a method of    preparing a protein having an activity of transferring an aromatic    acyl group to a sugar residue of a flavonoid using    1-O-acyl-β-D-glucose as an acyl donor or a protein having an    activity of transferring a glucosyl group to a hydroxyl group at    position 1 of hydroxycinnamic acid using UDP-glucose as a glucosyl    donor, which comprises culturing or growing the host cell of [9]    above and recovering the protein from the host cell.    [12] The 12th aspect of the present invention relates to a method of    preparing a protein by in vitro translation using the gene of any    one of [1] to [6] above.    [13] The 13th aspect of the present invention relates to a plant    which has been transformed by introducing thereinto the gene of any    one of [1] to [6] above or the vector of [7] or [8] above.    [14] The 14th aspect of the present invention relates to a offspring    of the plant of [13] above, which has the same nature as that of the    plant.    [15] The 15th aspect of the present invention relates to a tissue of    the plant of [13] above or the offspring of [14] above.    [16] The 16th aspect of the present invention relates to a cut    flower of the plant of [13] above or the offspring of [14] above.    [17] The 17th aspect of the present invention relates to a method of    transferring an aromatic acyl group to a sugar residue of a    flavonoid using 1-O-acyl-β-D-glucose as an acyl donor, which    comprises introducing the gene of any one of [1] to [3] above or the    vector of [7] above into a plant or plant cell and expressing the    gene.    [18] The 18th aspect of the present invention relates to a method of    modifying the flower color of plant, comprising introducing the gene    of any one [1] to [6] above or the vector of [7] or [8] above into a    plant or plant cell and expressing the gene.    [19] The 19th aspect of the present invention relates to a method of    modifying the flower color of a plant body in a plant having the    gene of any one of [1] to [6] above, comprising inhibiting the    expression of the gene.

Hereinbelow, the present invention will be described in detail.

(1) Gene

(1-1) First Gene

The first gene of the present invention encodes a protein having anactivity of transferring an aromatic acyl group to a sugar residue of aflavonoid using 1-O-acyl-β-D-glucose as an acyl donor. As examples ofthe first gene of the present invention, the following genes (A) to (D)may be given.

(A) A gene encoding a protein having the amino acid sequence as shown inSEQ ID NO: 2, 4, 6, 8, 10 or 12 and having the above-describedacyltransferase activity.

The expression “having the amino acid sequence as shown in SEQ ID NO: 2,4, 6, 8, 10 or 12” used herein is intended to include not only a proteinconsisting of the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8,10 or 12 alone but also a protein which has a plurality of amino acidsadded to the N-terminus or C-terminus of the above protein. The numberof amino acids added is not particularly limited as long as the proteinretains the above-described acyltransferase activity Usually, the numberis within 400, preferably within 50.

(B) A gene encoding a protein having the amino acid sequence as shown inSEQ ID NO: 2, 4, 6, 8, 10 or 12 which has addition, deletion and/orsubstitution of one or plurality of amino acids; and yet having theabove-described acyltransferase activity.

Such a protein having the amino acid sequence with addition, deletionand/or substitution may be either a natural protein or artificialprotein. The number of amino acids added, deleted and/or substituted isnot particularly limited as long as the protein retains theabove-described acyltransferase activity. Usually, the number is within20, preferably within 5.

(C) A gene encoding a protein having an amino acid sequence showing aspecific homology to the amino acid sequence as shown in SEQ ID NO: 2,4, 6, 8, 10 or 12 and yet having the above-described acyltransferaseactivity.

The term “specific homology” used herein means usually 20% or morehomology, preferably 50% or more homology, more preferably 60% or morehomology, most preferably 70% or more homology.

(D) A gene which hybridizes to a part or the whole of a nucleic acidrepresented by the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5,7, 9 or 11 or a nucleic acid encoding the amino acid sequence as shownin SEQ ID NO: 2, 4, 6, 8, 10 or 12 under stringent conditions andencodes a protein having the above-described acyltransferase activity.

The term “a part of a nucleic acid” used herein means, for example, apart encoding 6 or more consecutive amino acids within the consensussequence region. The term “stringent conditions” used herein means thoseconditions under which specific hybridization alone takes place andnon-specific hybridization does not occur. For example, conditions suchas temperature 50° C. and salt concentration 5×SSC (or concentrationequivalent thereto) may be given. It should be noted that appropriatehybridization temperature varies depending on the nucleotide sequence,length, etc. of the nucleic acid. For example, when a DNA fragmentconsisting of 18 nucleotides encoding 6 amino acids is used as a probe,50° C. or less is preferable.

Examples of genes selected by such hybridization include natural genes,e.g., plant-derived genes, especially genes derived from butterfly pea,lobelia and gentian. Genes selected by hybridization may be either cDNAor genomic DNA.

Of the above genes, genes occurring in nature may be obtained, forexample, by screening cDNA library as described later in Examples. Genesnot occurring in nature may also be obtained by using site directedmutagenesis, PCR or the like.

(1-2) Second Gene

The second gene of the present invention is a gene derived frombutterfly pea or lobelia encoding a protein that has an activity oftransferring a glucosyl group to a hydroxyl group at position 1 ofhydroxycinnamic acid using UDP-glucose as a glucosyl donor andsynthesizes 1-O-acyl-β-D-glucose as an acyl donor of the enzyme thatcatalyzes a reaction transferring an aromatic acyl group to a sugarresidue of anthocyanin.

As examples of the second gene of the present invention, the followinggenes (E) to (H) may be given.

(E) A gene encoding a protein having the amino acid sequence as shown inSEQ ID NO: 14, 16 or 18 and having the above-describedglucosyltransferase activity.

The expression “having the amino acid sequence as shown in SEQ ID NO:14, 16 or 18” used herein is intended to include not only a proteinconsisting of the amino acid sequence as shown in SEQ ID NO: 14, 16 or18 alone but also a protein which has a plurality of amino acids addedto the N-terminus or C-terminus of the above protein. The number ofamino acids added is not particularly limited as long as the proteinretains the above-described glucosyltransferase activity Usually, thenumber is within 400, preferably within 50.

(F) A gene encoding a protein having the amino acid sequence as shown inSEQ ID NO: 14, 16 or 18 which has addition, deletion and/or substitutionof one or plurality of amino acids; and yet having the above-describedglucosyltransferase activity.

Such a protein having the amino acid sequence with addition, deletionand/or substitution may be either a natural protein or artificialprotein. The number of amino acids added, deleted and/or substituted isnot particularly limited as long as the protein retains theabove-described glucosyltransferase activity. Usually, the number iswithin 20, preferably within 5.

(G) A gene encoding a protein having an amino acid sequence showing aspecific homology to the amino acid sequence as shown in SEQ ID NO: 14,16 or 18 and yet having the above-described glucosyltransferaseactivity.

The term “specific homology” used herein means usually 20% or morehomology, preferably 50% or more homology, more preferably 60% or morehomology, most preferably 70% or more homology.

(H) A gene which hybridizes to a part or the whole of a nucleic acidrepresented by the nucleotide sequence as shown in SEQ ID NO: 13, 15 or17 or a nucleic acid encoding the amino acid sequence as shown in SEQ IDNO: 14, 16 or 18 under stringent conditions and encodes a protein havingthe above-described glucosyltransferase activity.

The term “a part of a nucleic acid” used herein means, for example, apart encoding 6 or more consecutive amino acids within the consensussequence region. The term “stringent conditions” used herein means thoseconditions under which specific hybridization alone takes place andnon-specific hybridization does not occur. For example, conditions suchas temperature 50° C. and salt concentration 5×SSC (or concentrationequivalent thereto) may be given. It should be noted that appropriatehybridization temperature varies depending on the nucleotide sequence,length, etc. of the nucleic acid. For example, when a DNA fragmentconsisting of 18 nucleotides encoding 6 amino acids is used as a probe,50° C. or less is preferable.

Examples of genes selected by such hybridization include natural genes,e.g., plant-derived genes, especially genes derived from butterfly pea,lobelia and gentian. Genes selected by hybridization may be either cDNAor genomic DNA.

Of the above genes, genes occurring in nature may be obtained, forexample, by screening cDNA library as described later in Examples. Genesnot occurring in nature may also be obtained by using site directedmutagenesis, PCR or the like.

(2) Vector

The vector of the present invention may be prepared by inserting thegene described in (1) above into a known expression vector.

The known expression vector to be used is not particularly limited aslong as it comprises an appropriate promoter, terminator, replicationorigin, etc. As the promoter, trc promoter, tac promoter, lac promoteror the like may be used when the gene is to be expressed in bacteria;glycelaldehyde 3-phosphate dehydrogenase promoter, PH05 promoter or thelike may be used when the gene is to be expressed in yeasts; amylasepromoter, trpC promoter or the like may be used when the gene is to beexpressed in filamentous fungi; and SV40 early promoter, SV40 latepromoter, polyhedrin promoter or the like may be used when the gene isto be expressed in animal cells.

(3) Transformed Host Cell

The transformed host cell of the present invention is a host celltransformed by the vector described in (2) above.

The host cell may be either a prokaryote or eukaryote. Examples ofprokaryotes which may be used as a host cell include, but are notlimited to, Escherichia coli and Bacillus subtilis. Examples ofeukaryotes which may be used as a host cell include, but are not limitedto, yeasts, filamentous fungi, and cultured cells of animals and plants.Examples of yeasts include, but are not limited to, Saccharomycescerevisiae, Pichia patoris, Pichia methanolica and Schizosaccharomycespombe. Examples of filamentous fungi include, but are not limited to,Aspergillus oryzae and Aspergillus niger. Examples of animal cellsinclude, but are not limited to, rodents such as mouse (Mus musculus),Chinese hamster (Cricetulus griseus); primates such as monkey and human(Homo sapiens); amphibians such as Xenopus laevis; insects such asBombyx mori, Spodoptera frugiperda and Drosophila melonogaster.

The method of transformation by the vector is not particularly limited.The transformation may be performed according to conventional methods.

(4) Protein

The protein encoded by the gene described in (1) above is also includedin the present invention. This protein may be prepared, for example, bythe method described in (5) below.

(5) Method of Preparation of Protein

The method of preparing a protein according to the present inventioncomprises culturing or growing the host cell described in (3) above andthen recovering from the host cell a protein having an activity oftransferring an aromatic acyl group to a sugar residue of a flavonoidusing 1-O-acyl-β-D-glucose as an acyl donor or a protein having anactivity of transferring a glucosyl group to a hydroxyl group atposition 1 of hydroxycinnamic acid using UDP-glucose as a glucosyl donorand synthesizing an acyl donor. Alternatively, the method may becharacterized by in vitro translation or the like. Culturing or growingthe host cell may be performed by methods suitable for the type of thehost cell. The recovery of the protein may be performed by conventionalmethods. For example, the protein may be recovered and purified from thecultured cells or medium by techniques such as filtration, centrifuge,cell disruption, gel filtration chromatography, ion exchangechromatography, affinity chromatography or hydrophobic chromatography,etc. Thus, the protein of interest may be obtained.

(6) Plant

The plant of the present invention is a plant which has been transformedby introducing thereinto the gene described (1) above or the vectordescribed in (2) above.

The target plant into which the gene or vector is to be introduced isnot particularly limited. For example, rose, chrysanthemum, cineraria,snapdragon, cyclamen, orchid, prairie gentian, freesia, gerbera,gladiolus, babies'-breath, Kalanchoe blossfeldiana, lily, pelargonium,geranium, petunia, tulip, lobelia, Torenia foumieri, rice, barley,wheat, rapeseed, potato, tomato, aspen, banana, eucalyptus, sweetpotato, soybean, alfalfa, lupine, corn, cauliflower, lobelia, apple,grape, peach, Japanese persimmon, plum and citrus may be enumerated.

(7) Offspring of Plant

The offspring of the plant described in (6) above is also included inthe present invention.

(8) Tissue of Plant, etc.

Cells, tissues and organs of the plant described in (6) above or theoffspring of the plant described in (7) above are also included in thepresent invention.

(9) Cut Flower of Plant, etc.

Cut flowers of the plant described in (6) above or the offspring of theplant described in (7) above are also included in the present invention.

(10) Method of Transfer of Aromatic Acyl Group

A method of transferring an aromatic acyl group to a sugar residue of aflavonoid using 1-O-acyl-β-D-glucose as an acyl donor, which comprisesintroducing the gene described in (1-1) above or the vector described in(2) above (comprising the first gene) into a plant or plant cell andexpressing the gene, is also included in the present invention.

(11) Method of Modification of Flower Color

The method of modifying a flower color according to the presentinvention is a method of modifying the color of flower or fruit of aplant, comprising introducing the gene described in (1) above or thevector described in (2) above into a plant or plant cell and expressingthe gene; or inhibiting the expression of the gene described in (1)above in a plant having the gene. The target plant for gene transfer isnot particularly limited. For example, the plants enumerated in (6)above may be used. The target plant in which expression of the genedescribed in (1) is to be inhibited is not particularly limited as longas the plant has the gene.

Transfer and expression of the gene described in (1) above may beperformed by conventional methods. Inhibition of the expression of thegene described in (1) above may also be performed by conventionalmethods (e.g., antisense method, co-suppression method or RNAi method).

Effect of the Invention

By using the expression product of the gene obtained by the presentinvention, it is possible to transfer an aromatic acyl group to a sugarresidue of a flavonoid using 1-O-acyl-β-D-glucose as an acyl donor. As aresult, it has become possible to modify plant tissues (such as flowerand fruit) which are expressing colors via accumulation of flavonoids.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described in more detail withreference to the following Examples. The invention of the present patentapplication is not limited by the following Examples. Analyticalchemical techniques, molecular biological techniques and biochemicaltechniques were according to those described in Japanese UnexaminedPatent Publication No. 2005-95005 unless otherwise specified.

Example 1 Preparation of Substrates for Enzyme Reaction and StandardCompounds

(1) Preparation of Plant Materials

Seeds of butterfly pea (Clitoria ternatea) cv. Double blue (Sakata SeedCorporation) were sown aseptically and rooted, and then seedlings wereraised and grown in a glass greenhouse by conventional methods. Withrespect to Gentiana triflora cv. Hakkoda, cut flowers were collectedfrom plants cultivated in Aomori Prefecture and petals alone wereprepared for experiments. Seeds of Lobelia erinus cv. Riviera MidnightBlue, L. erinus cv. Aqua White, L. erinus cv. Aqua Lavender and L.erinus cv. Aqua Blue (all from Takii Seed) were sown in glassgreenhouses and then grown up to flowering in plastic pots. Flowerpetals and flower bud petals were collected and frozen instantaneouslywith liquid nitrogen, followed by storage at −80° C.

(2) Isolation of Compounds

As authentic samples of 1-O-hydroxycinnamoyl-β-D-glucoses (Milkowski etal., (2000) FEBS Letters 486: 183-184; Bokern and Strack (1988) Planta174:101-105; Bokern et al., (1991) Planta 184: 261-270; and Bokem etal., (1992) Botanica Acta 105: 146-151), i.e.1-O-p-coumaroyl-β-D-glucose, 1-O-caffeoyl-β-D-glucose,1-O-feruloyl-β-D-glucose and 1-O-sinapoyl-β-D-glucose, compoundsreleased from Dr. Alfred Baumert and Prof Dieter Strack (Leibniz IPB,Halle, Germany) were used. 1-O-p-Coumaroyl-β-D-glucose was purified andisolated from petals of butterfly pea; and 1-O-caffeoyl-β-D-glucose and1-O-feruloyl-β-D-glucose were purified and isolated from petals oflobelia (Kazuma et al., (2005) Abstracts of Japan Society forBioscience, Biotechnology and Agrochemistry 2005 Annual Conference: 3).Delphinidin 3-(6-malonyl)glucoside 3′,5′-diglucoside (ternatin C5:Terahara et al., (1998) Journal of Natural Products 61: 1361-1367),delphinidin 3,3′,5′-triglucoside (preternatin C5: Terahara et al.,(1990) Phytochemistry 29: 3686-3687) and delphinidin3-(6-malonyl)glucoside-3′-glucoside (Kazuma et al., (2005) Chemistry &Biodiversity 1: 1762-1770) were purified and isolated from petals ofbutterfly pea.

Example 2 Standard Enzyme Activity Measurement

A reaction solution (20 μl) containing 100 mM phosphate buffer (pH 6.5),0.4 mM anthocyanin (4 μl), 0.5 mM 1-O-acyl-β-D-glucose (4 μl) and anenzyme solution (8 μl) was reacted at 30° C. The reaction was terminatedby adding 4 μl of 1M aqueous hydrochloric acid solution. After additionof 24 μl of 5% acetonitrile containing 0.05 M TFA, the reaction solutionwas filtered with Millex-LH filter (Millipore) and then analyzed by HPLCby feeding a 10 μl aliquot.

HPLC analysis of anthocyanin acyltransferase reaction products wereperformed with Develosil ODS-UG-5 column (3.0 i.d.×250 mm; NomuraChemical) at a column temperature of 35° C. Against an initial solventthat was 0.05 M trifluoroacetate (TFA)-containing 5% acetonitrile (MeCN)aqueous solution, 0.05 M TFA-containing 40% MeCN was added with a linearconcentration gradient from 14 to 86% (20 min) and the reaction productswere eluted at a flow rate of 0.5 ml/min. The reaction products weredetected with PDA and their molecular weights were confirmed byLC-MS/MS.

Example 3 Examination of Substrates to Be Used in Enzyme ActivityMeasurement

In order to detect an enzyme activity of transferring an aromatic acylgroup to the sugar residue of flavonoids using 1-O-acyl-β-D-glucose asan acyl donor, various types of 1-O-acyl-β-D-glucose and anthocyaninwere examined. Briefly, enzyme activities were measured using 35-70%ammonium sulfate precipitate fraction of the protein extracted frombutterfly pea petals as an enzyme solution, preternatin C5 as an acylreceptor, and various 1-O-acyl-β-D-glucoses as an acyl donor. As aresult, acyltransferase activity was detected in all of the1-O-acyl-β-D-glucoses tested. Specific activity was higher in thefollowing order: 1-O-sinapoyl-β-D-glucose, 1-O-feruloyl-β-D-glucose,1-O-p-coumaroyl-β-D-glucose and 1-O-caffeoyl-β-D-glucose. Further,enzyme activities were measured using 1-O-p-coumaroyl-β-D-glucose as anacyl donor and various types of anthocyanin as an acyl acceptor. As aresult, acyltransferase activity was detected in all of the anthocyaninstested (preternatin C5, ternatin C5 and delphinidin3-(6-malonyl)glucoside-3′-glucoside).

Example 4 Purification of Protein 3′AT Having Enzyme Activity ofTransferring Aromatic Acyl Group to Glucosyl Group at Position 3′ ofAnthocyanin

From butterfly pea petals, an acyltransferase (3′AT) that is a proteinhaving an activity of transferring p-coumaroyl to position 3 of glucosylgroup at position 3′ was purified using preternatin C5 and1-O-p-coumaroyl-β-D-glucose as substrates for enzyme activitymeasurement, based on the purification methods for a glucoseacyltransferase that biosynthesizes 1,2-di-O-acyl-β-D-glucose (Li etal., (1999) Plant Physiology 121: 453-460; Li and Steffens (2000)Proceedings of the National Academy of Sciences of the United States ofAmerica 97: 6902-6907; PCT/WO97/48811). Protein purification processeswere performed at 0-4° C. Purification was achieved by carrying outprotein extraction, ammonium sulfate fractionation, ion exchangechromatography using TSK gel DEAE-TOYOPEARL 650M (Tosoh), chromatographyusing concanavalin A (ConA)-agarose (Honen), chromatography using Mono PHR 5/20 (Amersham Bioscience) and ion exchange chromatography using MonoQ HR 5/5 (Amersham Bioscience) in this order. For column chromatographyusing TSK gel DEAE-TOYOPEARL 650M, Mono P and Mono Q, FPLC (Pharmacia)was used. For column chromatography using ConA-agarose, an open columnwas used.

(1) Preparation of Crude Enzyme Solution

Frozen petals (510.5 g) from butterfly pea were ground in a mortar witha pestle in the presence of liquid nitrogen. Then, after addition ofabout 1,000 ml of buffer A [100 mM Tris-HCl (pH 7.5), 5 mMdithiothreitol (DTT), 10 μM p-amidinophenyl methylsulfonyl fluoride(pAPMSF)], 5 g of polyvinylpolypyrrolidone (PVPP) and an appropriateamount of sea sand, they were ground further. An extract suspension wasprepared therefrom and centrifuged at 7,000 rpm for 15 min. Theresultant supernatant was filtered with quadruply layered gauze. To thesupernatant of the resultant filtrate, 800 g of Dowex 1×2 (100-200 mesh;Muromachi Chemical) was added. The mixture was left stationary for 15min and then filtered with a nylon mesh to thereby obtain a crude enzymesolution (1240 ml).

(2) Ammonium Sulfate Fractionation

The crude enzyme solution was subjected to salting out with 35%saturated ammonium sulfate for 30 min. Then, insoluble matters wereremoved by centrifuging at 7,000 rpm for 20 min. After further saltingout with 70% saturated ammonium sulfate overnight, the solution wascentrifuged at 7,000 rpm for 20 min to thereby obtain a proteinprecipitate. This precipitate was redissolved in buffer B [20 mMTris-HCl (pH 7.5), 1 mM DTT, 10 μM pAPMSF] and desalted with SephadexG-25 Fine column (70 mm×40 mm i.d.; Amersham Bioscience) equilibratedwith buffer B. Protein fractions (1598.4 mg/144 ml) were collected andsubjected to the following chromatographies.

(3) DEAE Anion Exchange FPLC

TSK gel DEAE-TOYOPEARL 650M (30 ml) was packed in a column (XK16/20, 180mm×16 mm i.d.) and equilibrated with buffer B. The enzyme solution wasapplied to the column and the protein was fractionated with a lineargradient of NaCl concentration changing from 0 mM to 200 mM in 45 min ata flow rate of 8 m/min. After measurement of acyltransferase activity ineach fraction, active fractions (840 ml) were collected and subjected tosalting out with 90% saturated ammonium sulfate overnight. The resultantsolution was centrifuged at 7,000 rpm for 20 min to thereby obtain aprotein precipitate. This precipitate was redissolved in 40 ml of bufferB. The thus dissolved protein solution was divided into 5 ml aliquotsand stored at −80° C. until subsequent purification.

(4) ConA Agarose Column Chromatography

The cryopreserved DEAE active fraction (5 ml) was dissolved and thendesalted with a gel-filtration column PD-10 (Amersham Bioscience)equilibrated with buffer C [50 mM HEPES-NaOH (pH 7.5), 10% glycerol, 0.2M KCl]. The desalted protein solution (7 ml) was concentrated to 0.5 mlby ultracentrifugal filtration. The resultant concentrated proteinsolution (0.5 ml) was applied to ConA agarose (4 ml) packed in a columnand equilibrated with buffer C. After application of the concentratedprotein solution, the column was washed with 4-bed volumes of buffer C(16 ml). The protein adsorbed onto ConA agarose was eluted with 3-bedvolumes of buffer D [50 mM HEPES-NaOH (pH 7.5), 10% glycerol, 0.2 M KCl,1 M α-D-methylglucoside] (12 ml). The eluate was poured into a dialysiscolumn SnakeSkin Dialysis Tubing MWCO 10,000 (PIERCE Biotechnology) anddialyzed with buffer E [25 mM piperazine-HCl (pH 5.5)] (3,000 ml)overnight. After further desalting with PD-10, centrifugal concentrationwith Amicon Ultra (molecular weight cut off 10,000; Millipore) wasperformed to thereby obtain 0.5 ml of a protein solution (0.5 mg/ml).

(5) Mono P FPLC

The thus obtained ConA active fraction (0.5 ml) was applied to a Mono Pcolumn equilibrated with buffer E, at a flow rate of 0.8 ml/min. Afterthe application of this protein, the column was washed with buffer E (6ml). The protein was eluted with a 1:10 (v/v) dilution of Polybuffer74-HCl (pH 4.0) (32 ml). The eluate was divided into 0.8 ml aliquots,which were fractionated in test tubes each containing 0.08 ml of 0.5 MHEPES-NaOH (pH 7.5) and 0.08 ml of glycerol. Then, active fractions werecollected (8.8 ml) and concentrated into 1.5 ml of a protein solution bycentrifugal concentration.

(6) Mono Q Strong Anion Exchange FPLC

The thus obtained Mono P active fraction (1.5 ml) was applied to Mono QHR5/5 column equilibrated with buffer F [10 mM Tris-HCl (pH 7.5), 1 mMDTT, 10 μM pAPMSF], at a flow rate of 1.0 m/min. The protein solutionwas fractionated by 1 ml with a linear concentration gradient of liquidB from 0% to 25% in 60 min using buffer F [10 mM Tris-HCl (pH 7.5), 1 mMDTT, 10 μM pAPMSF] as liquid A and buffer G [10 mM Tris-HCl (pH 7.5), 1mM DTT, 10 μM pAPMSF, 1M NaCl] as liquid B. Active fractions (6 ml) werecollected and concentrated into 0.1 ml of a protein solution (0.9 μg/ml)by centrifugal concentration.

The specific activity of the 3′AT protein was found to be 1492.6pkat/mg. Compared to the specific activity of the DEAE active fractionof 0.252 pkat/mg, this represents 5923-fold purification. Further, whensilver-staining was performed after active fractions were fractionatedby SDS-PAGE, a clear 30.8 kDa band and a thin 24.1 kDa band alone weredetected. It is reported that serine carboxypeptidase-likeacyltransferase (SCPL-AT) is a protein which functions as ahetero-tetramer composed of a 34 kDa and a 24 kDa polypeptides (Li etal., (1999) Plant Physiology 121: 453-460) or a hetero-dimer composed ofa 30 kDa and a 17 kDa polypeptides (Shirley and Chapple (2003) Journalof Biological Chemistry 278: 19870-19877). Thus, the analytical resultsby SDS-PAGE demonstrated that the 3′AT protein purified from butterflypea petals was sufficiently uniformly purified. It is reported thatSCPL-AT, like serine carboxypeptidase (SCPase), is modified by sugarchains, and the 3′AT protein binds to ConA resin. Therefore, it ishighly possible that the 3′AT protein is modified by sugar chains.Further, since silver-staining weakly stains those polypeptides withsugar chain modification, it was shown that the 24.1 kDa subunit may bemodified with sugar chains.

Example 5 Partial Purification of 3′5′AT Protein Having Sequential AcylTransfer Activity to Glucosyl Group at Positions 3′ and 5′ ofAnthocyanin

In the process of purification of 3′ AT protein, an activity wasdetected which acylates the glucosyl groups at positions 3′ and 5′ ofthe B ring of preternatin C5 in succession. Although 3′5′AT activity wasalso detected in the 3′AT active fraction after ammonium sulfatefractionation, DEAE anion exchange FPLC, ConA chromatography and Mono PFPLC, 3′5′AT activity was not detected in the 3′AT active fraction afterMono Q FPLC. Then, the inventors performed fractionation and partialpurification of an acyltransferase that is a protein having an activityof transferring a feruloyl group to position 6 of glucosyl group atposition 3′ of anthocyanin (3′ AT) and a 3′5′AT protein that is aprotein having activity to transferring a feruloyl group to position 6of both glucosyl groups at positions 3′ and 5′ of anthocyanin from the3′AT protein obtained, using preternatin C5 and 1-O-feruloyl-β-D-glucoseas substrates for enzyme activity measurement.

(1) Preparation of Crude Enzyme Solution and Ammonium SulfateFractionation

A fraction (50 ml) containing a protein (269.15 mg) which has both 3′ATand 3′5′AT activities was obtained from 101.4 g of butterfly pea petalsaccording to the methods described in (1) Preparation of Crude EnzymeSolution and (2) Ammonium Sulfate Fractionation in Example 3 withnecessary modifications.

(2) DEAE Anion Exchange FPLC

The enzyme solution was applied to TSK gel DEAE-TOYOPEARL 650M columnequilibrated with buffer B, and the protein was fractionated with alinear gradient of NaCl concentration from 0 mM to 200 mM in 360 min ata flow rate of 1 ml/min. The recovered 3′AT active fraction (54 ml) and3′5′AT active fraction (42 ml) were subjected to salting out with 90%saturated ammonium sulfate overnight and then centrifuged at 7,000 rpmfor 20 min to thereby obtain. The resultant protein precipitates wereredissolved by the method described in (3) in Example 3 with necessarymodifications and then desalted and concentrated by the method describedin (4) in Example 3 with necessary modifications.

(3) ConA Agarose Chromatography

Concentrated protein solutions of 3′AT and 3′5′AT, respectively, wereapplied to ConA agarose (5 ml) separately. Chromatography, dialysis anddesalting were performed as described in (4) in Example 3.

(4) Mono P FPLC

ConA active fraction was subjected to chromatography, desalting andconcentration according to the methods described in (5) in Example 3.The specific activity of 3′AT activity in the 3′AT active fraction was45.9 pkat/mg, which represents 100-fold purification compared to thespecific activity of 0.46 pkat/mg after ammonium sulfate fractionation.3′5′AT activity was also detected in the 3′AT active fraction, and thespecific activity thereof was 3.6 pkat/mg. On the other hand, thespecific activity of 3′5′AT activity in the 3′5′AT active fraction was9.6 pkat/mg, which represents 74-fold purification compared to thespecific activity of 0.13 pkat/mg after ammonium sulfate fractionation.3′AT activity was also detected in the 3′5′AT active fraction, and thespecific activity thereof was 4.1 pkat/mg.

Therefore, existence of the following two proteins was recognized inbutterfly pea petals: 3′AT having an acyl transfer activity to glucosylgroup at position 3′ of anthocyanin B ring and 3′5′AT having asequential acyl transfer activity to glucosyl groups at positions 3′ and5′ in succession.

Example 6 Determination of the Amino Acid Sequence of Anthocyanin 3′ATProtein

The 3′AT protein purified in Example 4 (approx. 65 ng) was fractionatedby SDS-PAGE and stained with PAGE Blue83 (CBB R-250; Daiichi PureChemicals). The stained bands (30.8 kDa and 24.1 kDa) were cut out.These samples were designated CTDCPQ-30 and CTDCPQ-24, respectively Theywere treated with trypsin-containing Tris buffer (pH 8.0) at 35° C. for20 hr. Subsequently, the total volume of the solution was subjected toreversed phase HPLC to separate fragment peptides. As a control, aportion of the gel without any band was cut out and treated in the samemanner. The conditions of HPLC separation of fragment peptides were asdescribed below. Briefly, as a column, Symmetry C18 3.5 μm (1.0×150 mm;Waters) was used. The flow rate was 50 μl/min. Solvent A was 0.1%TFA-containing 2% acetonitrile solution. Solvent B was 0.09%TFA-containing 90% acetonitrile solution. For the initial 6 min, theconcentration of solvent B was 0%; in the subsequent 5 min, theconcentration was raised to 10%; in the subsequent 75 min, theconcentration was raised to 50%; in the subsequent 5 min, theconcentration was raised to 100%; then, the concentration of solvent Bwas retained at 100% for 5 min. Detection was carried out at 210 nm and280 nm. Fractionation was performed by 50 μl.

Fraction No. 35 and No. 44+45 of CTDCPQ-30 and fraction No. 18+19 ofCTDCPQ-24 were subjected to determination of amino acid sequences.N-terminal amino acid sequences of individual fragment peptides wereanalyzed using Procise 494 cLC Protein Sequencing System. The determinedamino acid sequences are shown below.

(SEQ ID NOs: 19 and 20) CTDCPQ-30-T35: (R/K)WLIDHPK (SEQ ID NOs: 21 and22) CTDCPQ-30-T44+45: (R/K)ISFAHILER (SEQ ID NOs: 23 and 24)CTDCPQ-24-T18+19: (R/K)RPLYEXNTM

Example 7 Design of Primers for Amplifying SCPL-AT cDNA Fragment

Nucleotide sequences for genes encoding proteins that catalyze reactionsusing 1-O-acyl-β-D-glucose as an acyl donor are highly homologous tonucleotide sequences for genes encoding serine carboxypeptidase(SCPase). Proteins that catalyze reactions using 1-O-acyl-β-D-glucose asan acyl donor are designated SCPL-AT (Milkowski and Strack (2004)Phytochemistry 65: 517-524). Then, degenerate primers were designedbased on regions and their nucleotide sequences highly conserved inSCPase and SCPL-AT. In order to specify highly conserved regions and todesign primers, multiple alignment using CLUSTAL W program, Block Marker(blocks.fhcrc.org/blocks/) and CODEHOP (blocks.fhcrc.org/codehop.html)were used. Sequences used for multiple alignment were the amino acidsequences of NCBI/EMBL/DDBJ accession numbers AF242849, AF275313,AF248647, AY033947, AY383718 and X80836 (REGION: 12728.14326) andUniProt/Swiss-Prot accession numbers P07519, P08819 and P37890. Thesynthesized CODEHOP primers and degenerate primers are shown below.

(SEQ ID NO: 25) cdhp Fd: GGACCCCGTGATGATCTGGYTIAMIGG (SEQ ID NO: 26)cdhp Rv: CCGCAGAAGCAGGAGCAICCIGGICC (SEQ ID NO: 27) blockA Fd:AMIGGWGGICCTGGITGYWSIWS (SEQ ID NO: 28) blockB Fd: GAIWSICCIGYIGGIWSIGG(SEQ ID NO: 29) blockC Fd: RTIGSIGGIGAIWSITAYDSIGG (SEQ ID NO: 30)blockE Rv: RTCRTGRTCICCISWRWA (SEQ ID NO: 31) blockF Ry:GGYTTRTAYTCIGGIRCIGTRTGICC

Example 8 Cloning of Butterfly Pea SCPL-AT cDNA

(1) Preparation of RNA

Butterfly pea petals were divided into stages in terms of flower budlength by 5 mm. Briefly, flower bud lengths of 5-10 mm were regarded asstage 1; flower bud lengths of 10-15 mm were regarded as stage 2; flowerbud lengths of 15-20 mm were regarded as stage 3; flower bud lengths of20-25 mm were regarded as stage 4; flower bud lengths of 25-30 mm wereregarded as stage 5; flower bud lengths of 30-35 mm were regarded asstage 6; flower bud lengths of 35-40 mm were regarded as stage 7; flowerbud lengths of 40-45 mm were regarded as stage 8; flower bud lengths of45-50 mm were regarded as stage 9; and the flowering stage was regardedas stage 10. Total RNA was prepared from several hundred milligrams ofpetals of each stage using TRIzol (Invitrogen). From the thus preparedtotal RNA (50 μg), poly(A)⁺RNA was purified for each stage usingOligotex-dT30 super (Takara Bio) according to the method recommended bythe manufacturer to thereby prepare 15 μl of poly(A)⁺RNA solution.

(2) Amplification of cDNA Fragment by Degenerate RT-PCR and CloningThereof

Using the purified poly(A)⁺RNA solution (15 μl) as a template, asingle-strand cDNA was prepared with 1st strand cDNA synthesis kit(Amersham Bioscience) according to the method recommended by themanufacturer. The cDNAs of all stages thus synthesized were mixed inequal amounts to thereby prepare a template for PCR reaction. For PCRreaction, the CODEHOP primers and degenerate primers as shown in Example7 and NotI-d(T)₁₈ primer (Amersham Bioscience) were used.

First, PCR reaction was performed with various primer pairs selectedfrom the following: cdhp Fd and blockA Fd primers and three types ofreverse primers. Using 2 μl of single strand cDNA solution as atemplate, 2 μl each of forward primer and reverse primer, and 2 units ofExTaq polymerase (Takara Bio), a 50 μl reaction solution was preparedand PCR was performed according to the method recommended by themanufacturer. Primer pairs used were 1: cdhp Fd and NotI-d(T)₁₈, 2:blockA Fd and NotI-d(T)₁₈, 3: cdhp Fd and blockE Rv, 4: cdhp Fd andblockF Rv, 5: blockA Fd and blockE Rv; and 6: blockA Fd and blockF Rv.The concentrations of CODEHOP primers and degenerate primers wereadjusted to 50 μM, and the concentration of NotI-d(T)₁₈ primer wasadjusted to 10 μM. Thermal conditions of the PCR reaction were asfollows: 95° C. for 3 min, then 40 cycles of 95° C. for 30 sec, 55° C.for 45 sec and 72° C. for 80 sec, and finally 72° C. for 7 min. Theresultant reaction products were subjected to agarose gelelectrophoresis. Products of expected sizes obtained from reactions withthe above-described primer pairs 3 to 6 were recovered from gelfragments.

Using the PCR product (1 μl) obtained with the above-described primerpair 1 or 2 as a template, nested PCR was performed. Combinations offorward and reverse primers were 1: blockA Fd and blockF Rv, 2: cdhp Fdand blockF Rv, and 3: cdhp Fd and NotI-R21 (5′-TGGAAGAATTCGCGGCCGCAG-3′:SEQ ID NO: 32). Using 1 μl of the PCR product as a template, 1 μl eachof forward primer and reverse primer, and 1 unit of ExTaq polymerase(Takara Bio), a 25 μl reaction solution was prepared and PCR wasperformed according to the method recommended by the manufacturer. PCRproducts of expected sizes obtained from the reaction using theabove-described primer pair 1 and as a template the PCR product obtainedwith the primer pair of cdhp Fd and NotI-d(T)₁₈ were recovered from gelfractions.

Subsequently, PCR was performed with various primer pairs selected fromthe following: blockB Fd and blockC Fd primers and three types ofreverse primers. Using 2 μl of single strand cDNA as a template, 2 μleach of forward primer and reverse primer, and 2 units of LATaqpolymerase (Takara Bio), a 50 μl reaction solution was prepared andsubjected to PCR. Primer pairs were 1: blockB Fd and NotI-d(T)₁₈, 2:blockC Fd and NotI-d(T)₁₈, 3: blockB Fd and blockE Rv, 4: blockC Fd andblockE Rv, 5: blockB Fd and blockF Rv and 6: blockC Fd and blockF Rv.The concentrations of CODEHOP primers and degenerate primers wereadjusted to 50 μM, and the concentration of NotI-d(T)₁₈ primer wasadjusted to 10 μM. Thermal conditions of the PCR reaction were asfollows: 95° C. for 3 min, then 40 cycles of 95° C. for 30 sec, 50° C.for 45 sec and 72° C. for 80 sec, and finally 72° C. for 7 min. Theresultant reaction products were subjected to agarose gelelectrophoresis. Products of expected sizes obtained from reactions withthe above-described primer pairs 4, 5 and 6 were recovered from gelfractions.

Using the PCR product (1 μl) obtained with the above-described primerpair 1, 2 or 3 as a template, nested PCR was performed individually.Primer pairs used were 1: blockB Fd and blockE Rv, 2: blockC Fd andblockE Rv, 3: blockB Fd and blockF Rv and 4: blockC Fd and blockF Rv.Using 100 pmole each of forward primer and reverse primer and 1 unit ofLATaq polymerase (Takara Bio), a 50 μl reaction solution was preparedand PCR was performed according to the method recommended by themanufacturer. PCR products were subjected to agarose gel electrophoresisand products of expected sizes were recovered from gel fragments. Then,purified PCR products were subcloned into pGEM-T Easy vector (Promega),followed by determination of their nucleotide sequences. In order toestimate the gene products encoded by the resultant clones, BLAST search(blast.genome.jp/) was used. Further, after multiple alignment usingCLUSTAL X, molecular phylogenetic trees were created with TreeViewprogram, followed by estimation of the functions of the cDNAs.

The following primer pairs generated clones homologous to the SCPase orSCPL-AT of interest in the following cases: when cdhp Fd and blockE Rvor blockA Fd and blockE Rv were used in the 1st PCR; when blockA Fd andblockF Rv were used in nested PCR using the 1st PCR product obtainedwith cdhp Fd and NotI-d(T)₁₈ as a template; when blockC Fd andNotI-d(T)₁₈ were used; or when blockB Fd or blockC Fd was used incombination with blockE Rv or blockF Rv. Twenty-nine cDNA fragmentclones which were believed to encode SCPase or SCPL-AT were obtained.Analyses by multiple alignment and with molecular phylogenetic treesconfirmed existence of 4 types of SCPL clones. These butterfly pea SCPLclones were designated CtSCPL1, 2, 3 and 4, respectively Among all, cDNAfragment clones CtSCPL1 and CtSCPL4 were highly homologous to SCPL-ATand positioned in the same crade as that of known SCPL-AT when molecularphylogenetic trees were created.

(3) RACE of CtSCPL1 and CtSCPL4 cDNA Fragments

Total RNA was prepared from butterfly pea petals by a modified CTABmethod (Chang et al., (1993) Plant Molecular Biology Reporter; Mukai andYamamoto, Plant Cell Engineering Series 7, pp. 57-62). From the totalRNA (250 μg), poly(A)⁺RNA was purified using Oligotex-dT super accordingto the method recommended by the manufacturer. From approx. 480 ng ofthe thus purified poly(A)⁺RNA, Gene Racer Ready cDNA (GRR cDNA) wassynthesized using GeneRacer kit (Invitrogen) according to the methodrecommended by the manufacturer.

The thus synthesized cDNA was diluted at 1:3 to prepare a cDNA solution.Using this cDNA solution as a template, PCR was performed with GeneRacer5′ primer (5′-CGACTGGAGCACGAGGACACTGA-3′: SEQ ID NO: 33; Invitrogen) andCtSCPL1-R1 primer (5′-TACTGGAATGGGAATACCAGAGTAAG-3′: SEQ ID NO: 34) orCtSCPL1-R2 primer (5′-GGCATGGTGAACTAATGTCCAGTCAC-3′:SEQ ID NO: 35) eachof which is specific to an internal sequence of CtSCPL1 cDNA fragment.Further, PCR was performed with GeneRacer 5′ primer and CtSCPL4-R1primer (5′-GTGTCGACCCAGTCACAGTTTG-3′: SEQ ID NO: 36) or CtSCPL4-R2primer (5′-CTGATATAACCTCATTGTATGACTCC-3′: SEQ ID NO: 37) each of whichis specific to an internal sequence of CtSCPL4 cDNA fragment. Briefly,using the GRR cDNA as a templete, a primer pair of GeneRacer 5′ primer(30 pmole) and CtSCPL1-R1 (20 pmole) or a pair of GeneRacer 5′ primer(30 pmole) and CtSCPL1-R2 (20 pmole) and LATaq polymerase, PCR wasperformed in a 50 μl reaction solution according to the methodrecommended by the polymerase manufacturer. Thermal conditions of thePCR reaction were as follows: 94° C. for 3 min, then 30 cycles of 94° C.for 30 sec, 65° C. for 45 sec and 72° C. for 80 sec, and finally 72° C.for 7 min. Further, nested PCR was performed using the 1st PCR productas a template and GeneRacer 5′ nested primer in combination withCtSCPL1-R1, CtSCPL1-R2, CtSCPL4-R1 or CtSCPL4-R2. Briefly, PCR wasperformed in a 50 μl reaction solution in the same manner as in the 1stPCR using the 1st PCR product as a template and LATaq polymerase (TakaraBio) according to the method recommended by the manufacturer. Productsfrom the 1st PCR and nested PCR were fractionated by 0.8% agarose gelelectrophoresis. Bands of amplified products were cut out and the PCRproducts were recovered. The PCR products purified from gel wereTA-cloned into pGEM-T Easy vector. Several clones obtained were analyzedto thereby determine the 5′ terminal nucleotide sequences of CtSCPL1 andCtSCPL4 cDNA fragments, respectively.

The synthesized GRR cDNA was diluted at 1:3 to prepare a cDNA solution.Using this cDNA solution as a template, PCR was performed with GeneRacer3′ primer (5′-GCTGTCAACGATACGCTACGTAACG-3′: SEQ ID NO: 38; Invitrogen)or Gene Racer 3′ nested primer (Invitrogen;5′-CGCTACGTAACGGCATGACAGTG-3′: SEQ ID NO: 39) as a reverse primer andCtSCPL1-F1 primer (5′-TCATAAGGGAAGTATTGGTGAATGGC-3′: SEQ ID NO: 40) orCtSCPL1-F2 primer (5′-GTTTACCTTTCACGTCGGACATTCC-3′: SEQ ID NO: 41), eachof which is specific to an internal sequence of CtSCPL1 cDNA fragment,as a forward primer. Further, PCR was performed using as a forwardprimer CtSCPL4-F1 primer (5′-AGTGCACTACACATTCGTAAGG-3′: SEQ ID NO: 42)or CtSCPL4-F2 primer (5′-GTAAATGGCGTCGATGTACCC-3′: SEQ ID NO: 43) eachof which is specific to an internal sequence of CtSCPL4 cDNA fragment.PCR, cloning and sequencing were performed in the same manner asperformed in 5′RACE to thereby determine the 3′ terminal nucleotidesequences of CtSCPL1 and CtSCPL4 cDNA fragments, respectively

(4) Cloning of the Entire Protein-Encoding Region in CtSCPL1 cDNA

The inventors synthesized, as a forward primer, pE-CtSCPL1-F(5′-GACGACGACAAGATGACCATAGTAGAGTTCCTTCCTG-3′: SEQ ID NO: 44) whichcontains the initiation codon of the protein predicted from the 5′ and3′ terminal nucleotide sequences obtained by RACE and, as a reverseprimer, pE-CtSCPL1-R (5′-GAGGAGAAGCCCGGTTATTATAGAATGGATGCCAAGTTGG-3′:SEQ ID NO: 45) which contains the termination codon of the aboveprotein. With the single strand cDNA as a template, PCR was performed ina 50 μl reaction solution using each 20 pmole of pE-CtSCPL1-F andpE-CtSCPL1-R and LATaq polymerase according to the method recommended bythe polymerase manufacturer. Thermal conditions of the PCR reaction wereas follows: 95° C. for 3 min, then 30 cycles of 95° C. for 30 sec, 55°C. for 45 sec and 72° C. for 80 sec, and finally 72° C. for 7 min. Theresultant reaction products were fractionated by agarose gelelectrophoresis and the bands of amplified products were cut out.Purified PCR products were subcloned into pET30 Ek/LIC (Novagen).Several pET-CtSCPL1 clones obtained were analyzed to thereby determinethe nucleotide sequence of the entire protein-encoding region ofCtSCPL1. As a result, it was confirmed that CtSCPL1 includes the entireinternal amino acid sequence of the anthocyanin 3′AT protein obtained inExample 6. Further, the 6th cycle amino acid X which was not detected inCTDCPQ-24-T18+19:(R/K)RPLYEXNTM (SEQ ID NOS: 23 and 24) is N accordingto the amino acid sequence predicted from cDNA and it is believed thatthis amino acid is modified with sugar chains. The ORF of CtSCPL1 is1464 bp and encodes a polypeptide consisting of 487 amino acid residues.This has three glycosylation sites and a secretion signal at the Nterminal. The nucleotide sequence of CtSCPL1 is shown in SEQ ID NO: 1and the amino acid sequence deduced therefrom is shown in SEQ ID NO: 2.

(5) Screening of cDNA Library

Approximately 100,000 clones in the butterfly pea petal cDNA librarywere screened using CtSCPL1, 2, 3 and 4 cDNA fragment clones andArabidopsis thaliana SNG 1 and SNG 2 gene cDNAs as probes. Screening wasperformed for each probe with final washing conditions of 55° C.,0.1×SSC, 0.1% SDS. Finally, 14 positive clones were obtained. Of these,13 clones were CtSCPL1 and the remaining one clone was CtSCPL3. Thelongest clone in the CtSCPL1 positive clones was CtSCPLA1-8 consistingof 1740 bp. When compared to the clone obtained by 5′ RACE, CtSCPLA1-8lacks a sequence upstream of the initiation Met (SEQ ID NO: 46:ATTAAAAAAAAATG). The nucleotide sequence of the open reading frame inthe 13 positive clones including CtSCPLA1-8 was identical with the cloneobtained by RACE and pET-CtSCPL1.

Example 9 Expression of Recombinant CtSCPL1 Protein inBaculovirus-Insect Cell System

(1) Preparation of CtSCPL1 Recombinant Baculovirus

For expression of recombinant proteins in Baculovirus-insect cellsystems, BaculoDirect Baculovirus Expression Systems (BaculoDirectC-Term Expression Kit; Invitrogen) were used. The protein-encodingregion predicted from the nucleotide sequence of the clone obtained inExample 8 was amplified by PCR using a forward primer (CtSCPL1-DTOPO-Fd:5′-CACCATGGCAGCCTTCAGTTCAACTCATA-3′: SEQ ID NO: 47) and a reverse primer(CtSCPL1-Rv-C-Tag: 5′-TAGAATGGATGCCAAGTTGGTGTATG-3′: SEQ ID NO: 48).Using the single strand cDNA (1 μl) as a template, 20 pmole each offorward primer and reverse primer, and 2 units of Pyrobest Taqpolymerase (Takara Bio), PCR was performed in a 50 μl reaction solutionaccording to the method recommended by the manufacturer. Thermalconditions of the PCR reaction were as follows: 94° C. for 3 min. then30 cycles of 94° C. for 30 sec, 65° C. for 45 sec and 72° C. for 80 sec,and finally 72° C. for 7 min. The resultant PCR product was subclonedinto pENTR-D-TOPO vector (Invitrogen) according to the methodrecommended by the manufacturer. Resultant several pENTR-CtSCPL1 cloneswere analyzed to thereby confirm the nucleotide sequences thereof UsingLR Clonase (Invitrogen), CtSCPL1 was recombined from pENTR-CtSCPL1 intoBaculoDirect C-Term Linear DNA. The LR reaction product was transfectedinto Spodoptera frugiperda ovary cell-derived Sf9 cells usingCellfection. Removal of non-recombinant virus using ganciclovir, growthof recombinant virus and measurement of viral titer were performedaccording to the methods recommended by the manufacturer to therebyprepare 2-3×10⁷pfu/ml of recombinant CtSCPL1 virus.

(2) Expression of Recombinant CtSCPL1 Protein and Confirmation of EnzymeActivity

Sf9 cells monolayer-cultured in complete Grace medium or Sf900II-SFMserum free medium (Gibco) in 6-well plates (3×10⁶ cells) were infectedwith the recombinant virus at a multiplicity of infection (MOI) of 5-10.After a five-day culture at 28° C., the culture broth and the Sf9 cellswere recovered. The culture broth was concentrated by centrifugation.The cells were suspended in a buffer and sonicated. The resultantcentrifugal supernatant was concentrated by centrifugation in the samemanner as applied to the culture broth. Enzyme reaction was performedusing the thus concentrated protein solution, and the reaction productwas analyzed by HPLC and LC-MS.

When this protein was reacted with preternatin C5 or delphinidin3-(6-malonyl)glucoside-3′-glucoside and 1-O-feruloyl-β-D-glucose assubstrates, a reaction product was obtained which had a molecular massindicating that one feruloyl group was attached to preternatin C5. Thus,3′AT activity was confirmed. Further, when the protein was reactedternatin C5 and 1-O-p-coumaroyl-β-D-glucose as substrates, 3′AT activitywas also recognized. Thus, it was confirmed that CtSCPL1 cDNA isencoding a protein which has an enzyme activity of transferring an acylgroup to a glucosyl group in the B ring of anthocyanin using1-O-acyl-β-D-glucose as an acyl donor. No difference caused by differentmedia was observed in activity. No activity was recognized in therecombinant virus-uninfected Sf9 cell extract solution or culture broth.Further, since the enzyme activity was recognized in both the Sf9 cellextract solution and the culture broth, this enzyme was found to be asecretory protein.

Example 10 Preparation of CtSCPL4 Recombinant Baculovirus

The protein-coding region (open reading frame) predicted from thenucleotide sequence of the clone obtained in Example 8 was amplified byPCR using a forward primer (CtSCPL4-DTOPO-Fd:5′-CACCATGGCGAGGTTTAGTTCAAGTCTTG-3′: SEQ ID NO: 49) and a reverse primer(CtSCPL4-Rv-Stop: 5′-TTACAAAGGCCTTTTAGATATCCATCTCC-3′SEQ ID NO: 50). PCRwas performed in the same manner as in Example 9, and the resultant PCRproduct was subcloned into pENTR-D-TOPO vector according to the methodrecommended by the manufacturer. Resultant several pENTR-CtSCPL4 cloneswere analyzed to thereby confirm the entire nucleotide sequence ofCtSCPL4. The nucleotide sequence in the ORF of CtSCPL4 is shown SEQ IDNO: 3 and the amino acid sequence deduced therefrom is shown in SEQ IDNO: 4. The ORF of CtSCPL4 is 1410 bp and encodes a polypeptideconsisting of 469 amino acid residues. This has three sugar chainmodification sites and a secretion signal at the N terminal. CtSCPL4 has79.1% homology to CtSCPL1 at the amino acid level and is located mostadjacent to CtSCPL1 in SCPL-AT crade in molecular phylogenic analysis.Therefore, it is possible to say that, like CtSCPL1, CtSCPL4 is alsoencoding a protein which has an enzyme activity of transferring an acylgroup especially to a glucosyl group in the B ring of anthocyanin using1-O-acyl-β-D-glucose as an acyl donor. CtSCPL4 was recombined frompENTR-CtSCPL4 into BaculoDirect Secreted Linear DNA using BaculoDirectBaculovirus Expression Systems, and recombinant CtSCPL4 virus wasprepared according to the method recommended by the manufacturer.

Example 11 Cloning of Gentian SCPL-AT cDNA

(1) Amplification and Cloning of cDNA Fragment Using Degenerate RT-PCR

Gentian petals were divided into two stages, i.e., flower bud length 2.5mm or less and flower bud length 2.5-3.5 mm. Total RNA was prepared from1.5 g of petals of each stage using TRIzol (Invitrogen). Using 5 μg ofthe thus obtained total RNA as a template, a single strand cDNA wassynthesized with 1st strand cDNA synthesis kit (Amersham Bioscience)according to the method recommended by the manufacturer. The synthesizedcDNAs from both stages were mixed in equal amounts to thereby prepare atemplate for PCR. For PCR reaction, the CODEHOP primers, degenerateprimers and NotI-d(T)₁₈ primer described in Example 6 were used.Briefly, using 2 μl of the single strand cDNA as a template and 2 μleach of forward primer and reverse primer and 1 unit of LATaqpolymerase, PCR reaction was performed in a 50 μl reaction solution.Thermal conditions of the PCR reaction were as follows: 95° C. for 3min, then 35 cycles of 95° C. for 30 sec, 48° C. for 45 sec and 72° C.for 80 sec, and finally 72° C. for 7 min. The reaction products obtainedfrom nested PCR using the 1st PCR product as a template were agarose gelelectrophoresed. Gel fragments which have those sizes as expected fromthe primer pairs used were recovered. Purified PCR products weresubcloned into pGEM-T Easy vector, followed by determination ofnucleotide sequences thereof.

The following primer pairs generated homologous clones to SCPase orSCPL-AT of interest in the following cases: when blockA Fd and blockFRv, or blockC Fd and blockF Rv were used in the 1st PCR; when blockC Fdand blockF Rv were used in nested PCR using as a template the 1st PCRproduct obtained using blockA Fd and NotI-d(T)₁₈; when blockC Fd andblockE Rv, or blockC Fd and blockF Rv were used in nested PCR using as atemplate the 1st PCR product obtained using blockC Fd and NotI-d(T)₁₈.The number of cDNA fragment clones which were believed to encode SCPaseor SCPL-AT was 17. As a result of analysis by multiple alignment andwith molecular phylogenetic trees, existence of four SCPL clones wasconfirmed. These gentian SCPL clones were designated GentrSCPL1, 2, 3and 4, respectively. Among all, cDNAfragment clones GentrSCPL1 andGentrSCPL2 were highly homologous to SCPL-AT and positioned in the samecrade as that of known SCPL-AT when molecular phylogenetic trees werecreated.

(2) RACE of GentrSCPL1 and GentrSCPL2 cDNA Fragments

Poly(A)⁺RNA was purified from the total RNA (total 550 μg) prepared fromeach stage in (1) in Example 11, using Oligotex-dT30 super according tothe method recommended by the manufacturer. From approx. 210 ng of thepurified poly(A)⁺RNA, GRR cDNA was synthesized using GeneRacer kitaccording to the method recommended by the manufacturer. The synthesizedGRR cDNA was diluted at 1:3 to prepare a cDNA solution. Using this cDNAsolution as a template, PCR was performed with GeneRacer 5′ primer and areverse primer specific to an internal sequence of GentrSCPL1 cDNAfragment (GentrSCPL1-R1 primer: 5′-GCATAAACCGTTGCTTTGATCCGCC-3′: SEQ IDNO: 5′ or GentrSCPL1-R2 primer: 5′-CATCAATGAAGCCATCAGCCACAGG-3′: SEQ IDNO: 52). Further, PCR was performed with GeneRacer 5′ primer and areverse primer specific to an internal sequence of GentrSCPL2 cDNAfragment (GentrSCPL2-R1 primer: 5′-TTAAGCACGTCAGGAATCCGGAGG-3′: SEQ IDNO: 53 or GentrSCPL2-R2 primer: 5′-TGAACGTCGAATGCCGTGAAACACC-3′: SEQ IDNO: 54). Briefly, using GGR cDNA (as a template), GeneRacer 5′ primer(30 pmole), a reverse primer (20 pmole) and LATaq polymerase, PCR wasperformed in a 50 μl reaction solution according to the methodrecommended by the polymerase manufacturer. Thermal conditions of thePCR reaction were as follows: 94° C. for 3 min, then 30 cycles of 94° C.for 30 sec, 65° C. for 45 sec and 72° C. for 80 sec, and finally 72° C.for 7 min. Further, nested PCR was performed using the 1st PCR productas a template and GeneRacer 5′ nested primer in combination with areverse primer. The 1st PCR and nested PCR products were fractionated byagarose gel electrophoresis. The bands of amplified products were cutout to thereby recover PCR products. The PCR products purified from gelwere subjected to TA cloning. Resultant several clones were analyzed tothereby determine the 5′ terminal nucleotide sequences of GentrSCPL1 andGentrSCPL2 cDNA fragments, respectively.

Using a 1:3 dilution of the synthesized GRR cDNA as a template, PCR wasperformed with GeneRacer 3′ primer or GeneRacer 3′ nested primer as areverse primer and GentrSCPL1-F1 primer (5′-TGGCATACAGTGGCGACCATGATC-3′:SEQ ID NO: 55) or GentrSCPL1-F2 primer (5′-CTGATGAGTGGCGTCCATGGAAAG-3′:SEQ ID NO: 56) each of which is specific to an internal sequence ofGentrSCPL1 cDNA fragment, as a forward primer. Further, PCR wasperformed using as a forward primer GentrSCPL2-F1 primer(5′-CGTTGTAACCGTTCGTTGCCATTCG-3′: SEQ ID NO: 57) or GentrSCPL2-F2 primer(5′-CGATGGTGCCATTCATGGCTACTC-3′: SEQ ID NO: 58) each of which isspecific to an internal sequence of GentrSCPL2 cDNA fragment. PCR,cloning and sequencing were performed in the same manner as in 5′RACE tothereby determine the 3′ terminal nucleotide sequences of GentrSCPL1 andGentrSCPL2 cDNA fragments, respectively.

(3) Cloning of Entire Protein-Encoding Region in GentrSCPL1 andGentrSCPL2 cDNA Fragments

The inventors synthesized forward primers containing the initiationcodon of the protein predicted from the 5′ and 3′ terminal nucleotidesequences obtained by RACE (Gentr1-DTOPO-F:5′-CACCATGGCGGTGCCGGCGGTGCC-3′: SEQ ID NO: 59 and Gentr2-DTOPO-F:5′-CACCATGGCGGATACAAACGGCACAGCC-3′: SEQ ID NO: 60) and reverse primerscontaining the predicted termination codon (Gentr1-Rv-CTag:5′-CAATGGAGAATCCGAGAAAAACCG-3′: SEQ ID NO: 61, Gentr1-Rv-Stop:5′-TTACAATGGAGAATCCGAGAAAAACCG-3′: SEQ ID NO: 62, Gentr2-Rv-CTag:5′-CAACGGTTTATGAGTTATCCACC-3′: SEQ ID NO: 63 and Gentr2-Rv-Stop:5′-CTACAACGGTTTATGAGTTATCCAC-3′: SEQ ID NO: 64). Using 1 μl of thesingle strand cDNA as a template, 20 pmole each of a forward primer anda reverse primer, and 2 units of Pyrobest Taq polymerase, PCR wasperformed in a 50 μl reaction solution according to the methodrecommended by the polymerase manufacturer. Thermal conditions of thePCR reaction were as follows: 94° C. for 3 min, then 30 cycles of 94° C.for 30 sec, 65° C. for 45 sec and 72° C. for 80 sec, and finally 72° C.for 7 min. The PCR products were subcloned into pENTR-D-TOPO vectoraccording to the method recommended by the manufacturer. Resultantseveral pENTR-GentrSCPL1 and pENTR-GentrSCPL2 clones were analyzed toconfirm nucleotide sequences thereof. Two ORFs were confirmed inGentrSCPL2 and designated GentrSCPL2-1 and GentrSCPL2-2, respectivelyThe nucleotide sequences in the ORFs in GentrSCPL1, GentrSCPL2-1 andGentrSCPL2-2 are shown in SEQ ID NOS: 5, 7 and 9, respectively. Theamino acid sequences deduced therefrom are shown in SEQ ID NOS: 6, 8 and10. The sizes of ORFs in GentrSCPL1, GentrSCPL2-1 and GentrSCPL2-2 were1446 bp, 1485 bp and 1455 bp, respectively. They were encodingpolypeptides consisting of 481, 494 and 484 amino acid residues,respectively. A secretion signal sequence was present in the N-terminalin GentrSCPL1, GentrSCPL2-1 and GentrSCPL2-2. They had 2, 3 and 3glycosylation sites, respectively, in their active protein codingregion. It was found that GentrSCPL2-2 is a clone where amino acids fromposition 226 to 235 of GentrSCPL2-1 are missing. GentrSCPL2-1 andGentrSCPL2-2 have 41% homology to CtSCPL1 at the amino acid level. Itwas also found in the molecular phylogenic analysis that they arepositioned adjacent to CtSCPL1, next to CtSCPL4, in SCPL-AT crade.Therefore, it is possible to describe that, like CtSCPL1, GentrSCPL2-1and GentrSCPL2-2 are encoding a protein which has an enzyme activity oftransferring an acyl group especially to a glucosyl group in the B ringof anthocyanin using 1-O-acyl-β-D-glucose as an acyl donor. GentrSCPL1is also positioned in the SCPL-AT grade and has 32% homology toGentrSCPL2 at the amino acid level. Thus, it is possible to describethat GentrSCPL1 is encoding an acyltransferase which uses1-O-acyl-β-D-glucose as an acyl donor. GentrSCPL1 and GentrSCPL2-1 wererecombined from pENTR-GentrSCPL1 and pENTR-GentrSCPL2-1 intoBaculoDirect C-Tag Linear DNA and BaculoDirect Secreted Linear DNA usingBaculoDirect Baculovirus Expression Systems, followed by preparation ofrecombinant GentrSCPL1 virus and recombinant GentrSCPL2-1 virusaccording to the method recommended by the manufacturer.

Example 12 Cloning of Lobelia SCPL-AT cDNA

(1) Amplification of cDNA Fragment by Degenerate PCR and Cloning

Poly(A)⁺RNA was prepared from petals of lobelia (Lobelia erinus cv.Riviera Midnight Blue) using QuickPrep Micro mRNA Purification Kit(Amersham Bioscience) according to the method recommended by themanufacturer. Using the resultant poly(A)⁺RNA as a template, singlestrand cDNA was synthesized with 1st strand cDNA synthesis kit accordingto the method recommended by the manufacturer. For the PCR reaction, theCODEHOP primers, degenerate primers and NotI-d(T)₁₈ primer described inExample 6 were used. Briefly, using 2 μl of the single strand cDNA as atemplate, 2 μl each of a forward primer and a reverse primer, and 1 unitof ExTaq polymerase, PCR was performed in a 50 μl reaction solution.Thermal conditions of the PCR reaction were as follows: 95° C. for 3min, then 30 cycles of 95° C. for 30 sec, 50° C. for 45 sec and 72° C.for 90 sec, and finally 72° C. for 7 min. PCR products obtained bynested PCR using 1st PCR products as templates were agarose gelelectrophoresed. Gel fragments with those sizes as expected fromindividual primer pairs used were recovered. Purified PCR products weresubcloned into pGEM-T Easy vector to thereby determine nucleotidesequences thereof.

The following primer pairs generated homologous clones to SCPase orSCPL-AT of interest in the following cases: when blockA Fd and blockFRv, blockC Fd and blockF Rv, or blockC Fd and NotI-d(T)₁₈ were used inthe 1st PCR, and when blockA Fd and blockF Rv, blockB Fd and blockE Rv,blockC Fd and blockE Rv, or blockC Fd and blockF Rv were used in nestedPCR. The number of cDNA fragment clones which were believed to encodeSCPase or SCPL-AT was 21. Among those clones, existence of LeSCPL1 (aSCPL-AT clone positioned in the same crade as that of known SCPL-AT) wasconfirmed.

(2) RACE of LeSCPL1 cDNA

Total RNA was prepared from 5 g of lobelia petals (2 g of floweredpetals and 3 g of flower bud petals) by a modified CTAB method. From theresultant total RNA, poly(A)⁺RNA was purified using Oligotex-dT30 superaccording to the method recommended by the manufacturer. GRR cDNA wassynthesized from approx. 480 ng of the purified poly(A)⁺RNA usingGeneRacer kit according to the method recommended by the manufacturer.

The synthesized GRR cDNA was diluted at 1:3 to prepare a cDNA solution.Using this cDNA solution as a template, PCR was performed with GeneRacer5′ primer and a reverse primer specific to an internal sequence ofLeSCPL1 cDNA fragment (LeSCPL1-R1 primer:5′-AATGGGTTGCCTAGCACGTATCCC-3′: SEQ ID NO: 65 or LeSCPL1-R2 primer:5′-GATTCGTGTTTGGCATCTGTCCAGC-3′: SEQ ID NO: 66). Briefly, using the GRRcDNA as a template, 30 pmole of GeneRacer 5′ primer, 20 pmole of areverse primer, and LATaq polymerase, PCR was performed in a 50 μlreaction solution. Thermal conditions of the PCR reaction were asfollows: 94° C. for 3 min, then 30 cycles of 94° C. for 30 sec, 65° C.for 45 sec and 72° C. for 80 sec, and finally 72° C. for 7 min. Further,using the 1st PCR product as a template, nested PCR was performed with acombination of GeneRacer 5′ nested primer and a reverse primer. The 1stPCR and nested PCR products were fractionated by agarose gelelectrophoresis, and the bands of amplified products were cut out tothereby recover the PCR products. The PCR products purified from gelwere TA-cloned. Resultant several clones were analyzed to determine the5′ terminal nucleotide sequence of the LeSCPL1.

Using a 1:3 dilution of the synthesized GRR cDNA as a template, PCR wasperformed with GeneRacer 3′ primer or GeneRacer 3′ nested primer as areverse primer and LeSCPL1-F1 primer (5′-AACGAGCCAGTTGTCCAACAAGCC-3′:SEQ ID NO: 67) or LeSCPL1-F2 primer (5′-CTCCACGTACGAAAGGGAACACTAAC-3′:SEQ ID NO: 68), each of which is specific to an internal sequence ofLeSCPL1 cDNA fragment, as a forward primer. PCR, cloning and sequencingwere performed in the same manner as in 5′RACE to thereby determine the3′ terminal nucleotide sequence of LeSCPL1 cDNA.

(3) Cloning of the Entire Protein-Encoding Region in LeSCPL1 cDNA

The inventors synthesized a forward primer which contains the initiationcodon of the protein predicted from the 5′ and 3′ terminal nucleotidesequences obtained by RACE (LeSCPL-DTOPO-F:5′-CACCATGGCGTTTGGTATGCCATTTTCG-3′: SEQ ID NO: 69) and reverse primerswhich contain the termination codon of the above protein(LeSCPL-Rv-CTag: 5′-CAATAAACTACGAGTAAGCCACCTTC-3′: SEQ ID NO: 70 andLeSCPL-Rv-Stop: 5′-TCACAATAAACTACGAGTAAGCCAC-3′: SEQ ID NO: 71). Using 1μl of the single strand cDNA as a template, 20 pmole each of a forwardprimer and a reverse primer, and 2 units of Pyrobest Taq polymerase(Takara Bio), PCR was performed in a 50 μl reaction solution accordingto the method recommended by the manufacturer. The resultant PCRproducts were subcloned into pENTR-D-TOPO vector according to the methodrecommended by the manufacturer. Resultant several pENTR-LeSCPL1 cloneswere analyzed to confirm the nucleotide sequences thereof. Thenucleotide sequence of the ORF of LeSCPL1 is shown in SEQ ID NO: 11, andthe amino acid sequence deduced therefrom is shown in SEQ ID NO: 12. TheORF of LeSCPL1 is 1466 bp, encoding a polypeptide consisting of 481amino acid residues. It contains 3 glycosylation sites. LeSCPL1 has 26%homology to CtSCPL1 at the amino acid level and is positioned in theSCPL-AT crade in molecular phylogenic analysis. Therefore, it ispossible to describe that LeSCPL1 is encoding an acyltransferase whichuses 1-O-acyl-p-D-glucose as an acyl donor. LeSCPL1 was recombined frompENTR-LeSCPL1 into BaculoDirect C-Tag Linear DNA and BaculoDirectSecreted Linear DNA using BaculoDirect Baculovirus Expression Systems.Then, recombinant virus was prepared according to the method recommendedby the manufacturer.

Example 13 Cloning of 1-O-Acyl-β-D-Glucose Synthase(UDP-Glucose:Hydroxycinnamate 1O-Glucosyltransferase) cDNA

(1) Isolation of Butterfly Pea 1-O-Acyl-β-D-Glucose Synthase cDNA

A degenerate primer GT-SPF (5′-WCICAYTGYGGITGGAAYTC-3′: SEQ ID NO: 72)was synthesized based on the amino acid sequence of PSPG-box (Huges andHuges (1994) DNA Seq., 5: 41-49), a region highly conserved in plantsecondary metabolite glucosyltransferases. Using single strand cDNA as atemplate, 100 pmole of Gt-SPF primer, 14 pmole of NotI-d(T)₁₈ primer and1 unit of ExTaq polymerase, PCR was performed in a 50 μl reactionsolution. Thermal conditions of the PCR reaction were as follows: 94° C.for 5 min, then 38 cycles of 94° C. for 30 sec, 42° C. for 30 sec and72° C. for 60 sec, and finally 72° C. for 10 min. The resultant PCRproduct was subcloned into pGEM-T easy vector according to the methodrecommended by the manufacturer. Several resultant clones were analyzedto confirm the nucleotide sequences thereof. As a result, a cDNAfragment clone GTC600-11 was obtained which shows high homology toUDP-glucose:hydroxycinnamate 1-O-glucosyltransferase (NCBI/EMBL/DDBJAccession No. AF287143; PIR Accession Nos. D71419, E71419 and F71419)found in plants such as Brassica napus and Arabidopsis thaliana. Usingthis cDNA fragment as a probe, 250,000 clones in butterfly pea petalcDNA library were screened with washing conditions of 2×SSC, 1% SDS and60° C. Finally, 7 clones were obtained in which the size of insertsubcloned into pBluescript SK- is 1.5 kbp or more. The predicted aminoacid sequences encoded by the ORFs of these clones were found identical;the longest clone containing the initiation codon was designatedCtGT11-4. The screening of the library was performed by known methods(see, for example, Japanese Unexamined Patent Publication No.2005-95005). CtGT11-4 gene was 1788 bp, encoding a polypeptideconsisting of 473 amino acid residues. The nucleotide sequence of thisgene is shown in SEQ ID NO: 13, and the deduced amino acid sequencetherefrom is shown in SEQ ID NO: 14.

(2) Confirmation of 1-O-Acyl-β-D-Glucose Synthase Activity in CtGT11-4Gene Product

For expressing CtGT11-4 gene, pET30Ek/LIC System was used. First, PCRwas performed using the following primers for amplifying the ORF cDNA ofCtGT11-4: pEGTC11-4F (5′-GACGACGACAAGATGGGGTCTGAAGCTTCGTTTC-3′: SEQ IDNO: 73) and pETGTC11-4R (5′-GAGGAGAAGCCCGGTCTAAGGGTTACCACGGTTTC-3′: SEQID NO: 74). Briefly, using the plasmid obtained in (1) in Example 12 asa template, 40 pmole each of pEGTC11-4F and pETGTC 11-4R, and 1 unit ofExTaq polymerase, PCR was performed in a 50 μl reaction solutionaccording to the method recommended by the manufacturer. The resultantPCR product was subcloned into pET30Ek/LIC vector according to themethod recommended by the manufacturer. Resultant several clones wereanalyzed to confirm the nucleotide sequences thereof, followed bytransformation into Escherichia coli BL21-CodonPlus(DE3)-RP(Stratagene).

The transformed E. coli was shaking-cultured overnight in 3 ml of LBmedium containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol. Thisculture broth (500 μl) was inoculated into LB medium (50 ml) andshaking-cultured until absorbance at 600 nm reached 0.4. Then,isopropyl-β-D-thiogalactoside (IPTG) was added to give a finalconcentration of 0.4 mM. The cells were shaking-cultured at 25° C. for16 hr and then harvested by refrigerated centrifugation (8000 rpm, 4°C., 20 min). CtGT11-4 protein was partially purified from the cellsusing Ni-NTA mini-column according to the method recommended by themanufacturer. Subsequently, the resultant protein was subjected tocentrifugal concentration with an ultrafilter and used in enzymeactivity measurement.

For measuring enzyme activity, a 30 μl reaction solution containing 100mM potassium phosphate buffer (pH 7.4), 30 pmole of UDP-glucose, 30pmole of hydroxycinnamic acid and 15 μl of recombinant protein solutionwas reacted at 30° C. (for 10 min, 20 min or 30 min), followed bytermination of the reaction by adding 6 μl of 1 M aqueous HCl solution.As hydroxycinnamic acid, p-coumaric acid, caffeic acid, ferulic acid andsinapic acid were used. The enzyme reaction products were analyzed byreversed-phase high performance liquid chromatography (ShiseidoNanoSpace system) using Develosil C30-UG-5 (1.5 i.d.×250 mm). Thesolvent retained a flow rate of 0.125 ml/min. Using 5% MeCN aqueoussolution as liquid A and 0.05 M TFA-containing 40% MeCN aqueous solutionas liquid B, a linear gradient was provided in such a manner that theconcentration of liquid B became 14% and 86% at 0 min and 20 min fromthe start of separation, respectively. The eluted materials weredetected with a PDA detector, and the resultant data were analyzed tothereby quantitatively determine 1-O-acyl-β-D-glucose.1-O-Hydroxycinnamoyl-β-D-glucoses (1-O-β-coumaroyl-β-D-glucose: Rt 8.1min; 1-O-caffeoyl-β-D-glucose: Rt 5.7 min; 1-O-feruloyl-β-D-glucose: Rt9.3 min; and 1-O-sinapoyl-β-D-glucose: Rt 9.8 min) were detected in thereaction products generated by recombinant CtGT11-4. As the reactiontime increased, the amounts of reaction products1-O-hydroxycinnamoyl-β-D-glucoses increased linearly. From theseresults, it was confirmed that CtGT11-4 gene is encoding an enzymehaving UDP-glucose:hydroxycinnamate 1-O-glucosyltransferase activityThus, it has become clear that CtGT11-4 gene is a 1-O-acyl-β-D-glucosesynthase gene.

(3) Isolation of Lobelia 1-O-Acyl-β-D-Glucose Synthase cDNA

According to the method described in (1) in Example 12, degenerateRT-PCR was performed using lobelia petal-derived single strand cDNA as atemplate. Then, cDNA fragments highly homologous to known genes werecloned. A cDNA library derived from petals of Lobelia erinus cv. RivieraMidnight Blue was constructed, and approx. 500,000 clones were screenedusing a cDNA fragment clone LeGT13 obtained above as a probe. Finally,28 positive clones were obtained. Predicted amino acid sequences encodedby their ORFs could be classified into two groups. The longest clones inthese two groups were designated LeGT13-20 and LeGT13-30, respectivelyLeGT13-20 and LeGT13-30 were 1574 bp and 1700 bp in size, respectively.They both had an initiation codon; their ORF was 1461 bp encoding apolypeptide consisting of 486 amino acid residues. Since LeGT13-20 andLeGT13-30 showed 95% homology to each other at the amino acid level, itwas believed that they are alleles encoding the same enzyme. Thenucleotide sequences of LeGT13-20 and LeGT13-30 are shown in SEQ ID NOs:15 and 17, and the amino acid sequences deduced therefrom are shown inSEQ ID NOs: 16 and 18.

(4) Confirmation of 1-O-Acyl-β-D-Glucose Synthase Activity in LeGT13-20and LeGT 13-30 Gene Products

LeGT13-20 and LeGT13-30 genes were expressed using pET30Ek/LIC System(Novagen). PCR was performed using the following primers for amplifyingthe ORF cDNA of LeGT13-20: pELeGT13A-F(5′-GACGACGACAAGATGGGCTCACTGCAGGGTACTACTACCGTC-3′ (SEQ ID NO: 75) andpELeGT13A-R (5′-GAGGAGAAGCCCGGTTAGTGCCCAACAACATCTTTTC-3′ (SEQ ID NO:76). Further, PCR was performed using the following primers foramplifying the ORF cDNA of LeGT13-30: pELeGT13B-F(5′-GACGACGACAAGATGGGCTCACTGCAGGGTACTACTACCGTT-3′ (SEQ ID NO: 77) andpELeGT13B-R (5′-GAGGAGAAGCCCGGTTAGTGCCCAATAACACCTTTTT-3′ (SEQ ID NO:78). Briefly, using the plasmid obtained in (3) in Example 12 as atemplate, 20 pmole of pELeGT13A-F or pELeGT13B-F as a forward primer, 20pmole of pELeGT13A-R or pELeGT13B-R as a reverse primer, and 1 unit ofExTaq polymerase, PCR was performed in a 50 μl reaction solutionaccording to the method recommended by the polymerase manufacturer. Theresultant PCR products were subcloned into pET30Ek/LIC vector accordingto the method recommended by the manufacturer. Resultant several cloneswere analyzed to confirm the nucleotide sequences thereof, and thentransformed into E. coli BL21-CodonPlus(DE3)-RP. Expression of thetransferred genes in transformed E. coli, partial purification of therecombinant proteins, and analysis of enzyme reaction and reactionproducts were performed in the same manner as described in (2) inExample 12. As a result, glucosyltransferase activity was confirmedagainst all of the four types of hydroxycinnamic acid used. From theseresults, it was confirmed that LeGT13-20 gene and LeGT13-30 gene areencoding an enzyme having UDP-glucose:hydroxycinnamate1-O-glucosyltransferase activity. Thus, it has become clear that bothLeGT13-20 gene and LeGT13-30 gene are a 1-O-acyl-β-D-glucose synthasegene.

1. An isolated gene encoding a protein having an activity oftransferring an aromatic acyl group to a sugar residue of a flavonoidusing 1-O-acyl-β-D-glucose as an acyl donor, wherein the gene comprises:(a) a nucleotide sequence selected from the group consisting of SEQ IDNOS: 1, 3, 5, 7, 9 and 10; (b) a nucleotide sequence encoding an aminoacid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6,8, 10 and 12; or (c) a nucleotide sequence encoding an amino acidsequence having 70% or more homology with any one of SEQ ID NOS: 2, 6,8, 10 and 12 or an amino acid sequence having at least 79.1% homologywith SEQ ID NO:4.
 2. A vector comprising the gene according to claim 1.3. A host cell which has been transformed by the vector according toclaim
 2. 4. A method of preparing a protein having an activity oftransferring an aromatic acyl group to a sugar residue of a flavonoidusing 1-O-acyl-β-D-glucose as an acyl donor or a protein having anactivity of transferring a glucosyl group to a hydroxyl group atposition 1 of hydroxycinnamic acid using UDP-glucose as a glucosyldonor, which comprises culturing or growing the host cell according toclaim 3 and recovering said protein from said host cell.
 5. A proteinencoded by the gene according to claim
 1. 6. A method of preparing aprotein by in vitro translation using the gene according to claim
 1. 7.A plant which has been transformed by introducing thereinto the geneaccording to claim 1 or a vector comprising the gene according to claim1 or an offspring of the plant.
 8. An offspring of the plant accordingto claim 7, wherein the offspring expresses the gene.
 9. A tissue of theplant or the offspring according to claim
 7. 10. A cut flower of theplant or the offspring according to claim
 7. 11. A method oftransferring an aromatic acyl group to a sugar residue of a flavonoidusing 1-O-acyl-β-D-glucose as an acyl donor, which comprises introducingthe gene according to claim 1 or a vector comprising the gene accordingto claim 1 into a plant or plant cell and expressing said gene.
 12. Amethod of modifying the flower color, comprising introducing the geneaccording to claim 1 or a vector comprising the gene according to claim1 into a plant or plant cell and expressing said gene.
 13. A method ofmodifying the flower color in a plant having the gene according to claim1, comprising inhibiting the expression of said gene.
 14. An isolatedgene encoding a protein having an activity of transferring an aromaticacyl group to a sugar residue of a flavonoid using 1-O-acyl-β-D-glucoseas an acyl donor, wherein the gene comprises: (a) a nucleotide sequenceselected from the group consisting of SEQ ID NOS: 1, 5, 7, 9 and 10; (b)a nucleotide sequence encoding an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 2, 6, 8, 10 and 12; or (c) a nucleotidesequence encoding an amino acid sequence having 70% or more homologywith any one of SEQ ID NOS: 2, 6, 8, 10 and 12.